衰变加速因子介导Jurkat细胞的PTK信号传递机制的实验研究

为研究衰变加速因子 (DAF )介导Jurkat细胞信号传递的机制 ,本文应用免疫印迹及化学发光技术 (ECL)发现交联DAF单抗可使CD3+ 与CD3 Jurkat细胞总蛋白酪氨酸磷酸化水平增加 ,但磷酸化水平不同。免疫共沉淀实验检测到DAF与Src家族酪氨酸激酶 (SrcPTK )可发生共沉淀反应。去污剂不溶膜复合物 (DIG )抽提与鉴定检测到DIG内有SrcPTK与DAF的特异性聚集。说明DAF对Jurkat细胞的信号传递有辅助激活效应 ,此效应可能是通过其以与膜微区相关的所联系的SrcPTK实现的     

衰变加速因子及其所在膜微区在T淋巴细胞信号传递中的作用

低温抽提Jurkat细胞膜去污剂抗性的膜成分(DIG),检测Src家族酪氨酸激酶(PTK)及衰变加速因子(DAF)的分布情况。共聚焦扫描显微技术检测Jurkat细胞静息与交联状态下DAF分子对去污剂Triton-X100的抗溶性,以探讨其在Jur-kat细胞免疫识别中的作用,结果显示静息状态CD3对去污剂无抗溶性,而DAF与Lck有抗溶性。单抗交联后CD3对去污剂抗性有所增加。静息状态DAF与Src家族PTK特异分布于膜微区中,交联CD3可诱导CD3与膜微区特异性聚集,DAF所在的膜微区可能促进T细胞的免疫识别和信号传递作用。     

衰变加速因子结构基因及调控

衰变加速因子DAF是补体激活调节剂(RCA)家族中重要成员之一,在体内的存在形式有3种:分泌型DAF、跨膜型DAF和GPI锚链型DAF。DAF的分子结构、基因结构、合成及基因表达调控等方面的进展近年颇为迅速。DAF分子的研究,使人们了解了在细胞表面控制补体活性的分子基础。DAF基因克隆表达及转基因动物研制成功,为DAF的研究和应用开辟了广阔的前景。     

虫草多糖对人外周血IL—2,IL—2R及IFN—r调节作用的研究

本研究观察了冬虫夏草多糖对体外培养的外周血淋巴细胞的ＩＬ－２、ＩＬ－２Ｒ、ＩＦＮ－ｒ的影响。结果表明，ＣＰ可单独或协同ＰＨＡ诱导ＩＬ－２Ｒ的表达，促进可溶性ＩＬ－２Ｒ的生成，但对ＰＨＡ诱生的ＩＬ－２、ＩＦＮ－ｒ活性有选择性抑制作用。其协同或抑制作用均呈剂量依赖性，吲哚美辛可部分或全部消除其抑制效应。提示ＣＰ对体外培养的ＰＢＬ具有双向免疫调节作用。     

枸杞多糖对衰老小鼠IL—2、IL—2R的效应

枸杞子是茄科落叶灌木植物宁夏枸杞 (ＬｙｃｉｕｍｂａｒｂａｒｕｍＬ )和枸杞 (Ｌ ｃｈｉｎｅｎｓｅＭｉｌｌ )的成熟干燥果实 ,具有滋补肝肾、益精明目之功。枸杞多糖 (ｌｙｃｉ ｕｍｂａｒｂａｒｕｍｐｏｌｙｓａｃｃｈａｒｉｄｅｓ ,ＬＢＰ)是枸杞子的主要活性成分。现代药理学研究发现 ,枸杞多糖具有广泛的药理学作用 :增强免疫 ,抗癌抑瘤 ,抗过氧化、抗衰老 ,促进造血等[1 4 ] 。近些年有关从枸杞多糖对ＩＬ 2影响来探讨其免疫调节机理的报道很多[1,5 7] ,但对ＩＬ 2Ｒ的研究则少见。本文采用老年小鼠和目前应用得较成熟的Ｄ 半乳糖致…     

人参四物汤对成瘾动物胸腺T细胞亚群增殖及IL—2R的作用研究

目的：观察吗啡对胸腺细胞ＣＤ４,ＣＤ８细胞周期增殖、ＩＬ－２的影响及人参四物汤对其调节作用。方法：小剂量递增给药建立吗啡成瘾动物模型后,采用流式细胞仪分析各组小鼠胸腺ＣＤ４、ＣＤ８细胞ＤＮＡ含量、增殖九以及ＩＬ－２Ｒ的情况。结果：吗啡成瘾组小鼠ＣＤ４、ＣＤ８细胞Ｓ,Ｇ２＋Ｍ期和增殖指数显著低于正常组和人参四物汤组动物,而Ｃ０／Ｃ１期高于后二者;吗啡依赖小鼠ＩＬ－２Ｒ表达下降,人参四物汽组动物的Ｔ细     

人参四物汤对成瘾动物胸腺T细胞亚群增殖及IL—2R的作用研究

目的：观察吗啡对胸腺细胞ＣＤ４,ＣＤ８细胞周期增殖、ＩＬ－２的影响及人参四物汤对其调节作用。方法：小剂量递增给药建立吗啡成瘾动物模型后,采用流式细胞仪分析各组小鼠胸腺ＣＤ４、ＣＤ８细胞ＤＮＡ含量、增殖九以及ＩＬ－２Ｒ的情况。结果：吗啡成瘾组小鼠ＣＤ４、ＣＤ８细胞Ｓ,Ｇ２＋Ｍ期和增殖指数显著低于正常组和人参四物汤组动物,而Ｃ０／Ｃ１期高于后二者;吗啡依赖小鼠ＩＬ－２Ｒ表达下降,人参四物汽组动物的Ｔ细     

Molecular cloning of murine decay accelerating factor by immunoscreening

Although the cDNA of human decay accelerating factor (DAF) whichrestricts complement activation on homologous cell membraneswas cloned in 1987, all trials to detect the cDNA of mouse DAFby cross-hybridization were unsuccessful. However, by immunoscreeningwith a rabbit antiserum against purified mouse DAF, we successfullycloned the cDNA. It contains four typical short consensus repeats(SCR) similar to that In human and guinea pig DAF. The basesequence showed 63.7 and 63.8% identIty to that of human andguinea pig DAF respectively. The deduced amino acid sequenceidentity to human and guinea pig DAF was 47.2 and 46.5% respectively.Mouse complement receptor related gene Y (Crry)/p65 functionis comparable to DAF. SCR3 and SCR4 of mouse DAF showed 50%identity to SCR2 and SCR3 of Crty/p65 respectively. IdentificatIonof the mouse DAF gene should open a new approach for determiningthe actual in vlvo role of DAF by analyzing autoimmune miceas well as generating DAF gene knockout mice using embryonicstem cells.     

酪氨酸磷酸化抑制剂AG490抑制Jurkat T细胞增殖

目的:通过AG490对Jurkat T细胞活化、增殖、周期、凋亡及ICBP90蛋白表达的影响,探讨阻断JAK/STAT信号通路以抑制Jurkat T细胞生长的可能性及其初步机制。方法:以Jurkat T细胞为模型,应用双荧光抗体标记结合流式细胞仪检测AG490对Jurkat T细胞表面分子CD69和CD25表达的影响;利用噻唑蓝(MTT)比色法观察AG490对Jurkat T细胞增殖的影响;采用碘化丙锭(PI)染色检测AG490对Jur-kat T细胞周期的影响;应用Annexin V-FITC和PI双染色检测AG490对Jurkat T细胞凋亡的影响;Western blot检测AG490对Jurkat T细胞中ICBP90蛋白表达的影响以确定其与AG490抑制Jurkat T细胞增殖的关系。结果:随着AG490浓度从1 mmol/L增至30 mmol/L,细胞停滞于G0/G1期,阻止其进入S期和G2/M期,导致Jurkat T细胞ICBP90蛋白的表达显著降低;AG490对细胞的抑制作用于24 h最为明显,抑制率可达27.37%,呈剂量依赖关系;AG490不能明显抑制Jurkat T细胞的活化或促进其凋亡。结论:AG490能明显抑制Jurkat T细胞的生长,其抑制作用可能通过下调Jurkat T细胞ICBP90蛋白的表达与细胞周期阻滞有关,而不是通过促进细胞凋亡而实现。     

IL-22 in the endometriotic milieu promotes the proliferation of endometrial stromal cells via stimulating the secretion of CCL2 and IL-8

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family and plays critical roles in inflammation, immune surveillance, and tissue homeostasis. However, whether IL-22 regulates the growth of endometrial stromal cells (ESCs), and participates in the pathogenesis of endometriosis remain unclear. In this study, we found that the expression of IL-22 and it receptors (IL-22R1 and IL-10R2) in eutopic endometrium and ectopic lesion of women with endometriosis was higher than that from healthy control. Recombinant human IL-22 (rhIL-22) stimulated the proliferation of ESCs in a dosage-dependent manner. On the contrary, anti-human IL-22 neutralizing antibody inhibited the proliferation of ESCs in vitro. The stimulatory effect of IL-22 on the proliferation of ESCs could be reversed by inhibitor of STAT5, ERK1/2 or AKT signal pathway. However, blocking STAT3, JNK or P38 signal pathway had no these effects. By Enzyme-linked immunosorbent assay (ELISA) and flow cytometry assay, we demonstrated the rhIL-22 not only stimulate the secretion of CCL2 and IL-8, but also significantly up-regulate the expression of IL-8 receptor CXCR1 on ESCs. Meanwhile, STAT5, ERK1/2 and or AKT signal inhibitors could abrogate the increase of CCL2, IL-8 and CXCR1 levels induced by rhIL-22. However, rhIL-22 had not similar influence on CCL2 receptor CCR2. Our current results suggested that the higher level of IL-22 and it receptors in eutopic endometrium may stimulate the expression of CCL2, IL-8/CXCR1, and further promote the growth of ESCs possibly through activating STAT5, MAPK/ERK1/2 and or AKT signal pathways, which may be involved in the occurrence and development of endometriosis.     

衰变加速因子在非小细胞肺癌中的表达及其临床意义

目的 检测衰变加速因子（decay accelerating factor,DAF,CD55）在非小细胞肺癌（non-small cell lung carcinoma,NSCLC）中的表达,分析其表达与临床分期、化疗用药及预后的关系。方法 免疫组化法检测8例NSCLC癌旁组织和36例NSCLC手术标本中DAF的蛋白表达,分析临床资料。结果 免疫组化结果显示36例NSCLC标本中,共有18例（50．0％）表达DAF,其中18例肺鳞癌中有15例（83．3％）表达,而16例肺腺癌中有3例（18．8％）表达,两者有显著差异（P〈0．05）;不同的病理分期的DAF免疫组化平均积分光密度表达有显著性差异（P〈0．05）;DAF的表达与无病生存期无相关关系（P〉0．05）。结论 NSCLC中有DAF的表达,尤其是肺鳞癌;提示在NSCLC抗体治疗中应同时阻断DAF的免疫抑制效应。     

胆固醇缺乏抑制Jurkat T淋巴细胞增殖及分子机制研究

人蛋白酪氨酸激酶信号传递系统及细胞周期调控这一角度来探讨胆固醇缺乏抑制Ｊｕｒｋａｔ细胞增殖的分子机制。方法采用接免疫荧光染色，流式细胞久仰分析技术及免疫细胞化学染色等方法做相关分析。结果经去脂血清培养基培养的Ｊｕｒｋａｔ细胞加ｌｏｖａｓｔａｔｉｎ处理３ｄ后，＾３Ｈ－ＴｄＲ掺入率明显下降，细胞增殖受抑细胞受阻于Ｇ０／Ｇ１期，ＰＴＫ活性及细胞ｃｙｃｌｉｎＤ１、ＣＤＫ４蛋白的表达明显降低，加入ＬＤＬ可以     

IL-4 enhances IEC-6 intestinal epithelial cell proliferation yet has no effect on IL-6 secretion

Intestinal epithelial cells (IEC) form an important line of defence at the intestinal mucosa by providing a barrier to lumenal contents and also by their ability to secrete various inflammatory cytokines. Recently, several T cell-derived cytokines have been shown to regulate specific IEC functions. In this study, the effect of IL-4 on IEC proliferation and secretion of the inflammatory cytokine IL-6 was investigated using the non-transformed rat IEC-6 intestinal epithelial cell line. Recombinant rat (rr)IL-4 was found to enhance IEC-6 cell proliferation over 4 days of culture, and this enhancement was dose-dependent. Further studies using specific antibodies confirmed that IL-4 induced the effect and that the effect was not mediated by autocrine-produced transforming growth factor-alpha. However, IL-4 did not induce IL-6 secretion by the IEC-6 cells, nor did it alter IL-1β-induced IL-6 secretion. These results indicate that T cells may be capable of regulating IEC proliferation via the secretion of IL-4 without altering the capacity of the IEC to function in the inflammatory response by secreting IL-6.     

人TRIM22基因真核表达质粒的构建和表达及TRIM22对Jurkat T细胞增殖的影响

研究TRIM22对Jurkat T增殖的影响,探讨TRIM22分子的生物学效应。分离人外周血单个核细胞,获得其细胞总RNA,采用RT-PCR方法扩增人TRIM22基因,经Nhe I和Hind III双酶切后插入pcDNA3.1/myc-His(-)真核表达载体,并对其进行酶切和测序鉴定。将构建好的真核表达质粒pcDNA/TRIM22转染Jurkat T细胞,用Western blot方法鉴定TRIM22蛋白表达情况,用CCK-8试剂检测TRIM22对T细胞增殖的影响,用双荧光素酶报告基因方法检测TRIM22对IL-2基因启动子的转录活性,用半定量RT-PCR方法检测TRIM22对IL-2 mRNA表达水平的影响。结果:成功构建了C端带有myc标记的真核表达质粒pcDNA/TRIM22,质粒转染Jurkat T细胞后能够表达TRIM22蛋白,细胞增殖检测结果显示TRIM22能够降低Jurkat T细胞的增殖达30%左右,报告基因检测结果显示TRIM22能够抑制IL-2启动子活性达20%至50%,过表达TRIM22降低IL-2 mRNA表达水平,这些结果为进一步深入研究TRIM22的生物学特性奠定了基础。